At least three distinct types of cyclic nucleotide phosphodiesterases (PDE) are associated, via different forces, with washed particulate fractions from bovine brain cortex. The detergent-solubilized, particulate cGMP-stimulated PDE was purified via cyclic nucleotide affinity chromatography and heparin-agarose, and exhibits in SDS-PAGE and Western immunoblots an Mr slightly greater than the soluble form of the enzyme isolated from calf liver or bovine brain. The purified particulate PDE is less sensitive to proteolysis by trypsin than the supernatant PDE. Our findings suggest the presence of at least two distinct isoenzymes of the cGMP-stimulated PDE "family" in bovine brain.